Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages

ABSTRACT

A polymer composition is disclosed composed of morpholino subunit structures linked together by uncharged, chiral linkages, one-three atoms in length. These chiral linkages join the morpholino nitrogen of one subunit to the 5&#39; exocyclic carbon of an adjacent subunit. Each subunit contains a purine or pyrimidine base-pairing moiety effective to bind by hydrogen bonding to a specific base or base-pair in a target polynucleotide.

This application is a continuation of application Ser. No. 454,057,filed Dec. 20, 1989 now abandoned, which is a continuation-in-part (CIP)of co-owned U.S. patent application Ser. No. 07/100,033 filed Sep. 23,1987, now U.S. Pat. No. 5,142,047, application Ser. No. 07/100,033 is aCIP of pending U.S. patent application Ser. No. 06/944,707, filed Dec.18, 1986, and a CIP of Ser. No. 06/911,258, filed Sep. 24, 1986, nowabandoned, and a CIP of Ser. No. 06/712,396 filed Mar. 15, 1985, nowabandoned.

This application was filed on even date with co-pending U.S. patentapplications Ser. Nos. 07/454,056 and 07/454,055, now issued as U.S.Pat. No. 5,034,506.

FIELD OF THE INVENTION

The present invention relates to morpholino-based polymers.

REFERENCES

Agarwal, Proc Nat Acad Sci USA, 85:7079 (1988) Balgobin, N., et al.,Tetrahedron Lett, 22:3667 (1981). Belikova, Tetrahedron Lett, 37:3557(1967). Blake et al., Biochem, 24:6132 (1985a). Blake et al., Biochem24:6139 (1985b). Bower et al., Nucleic Acids Res. 15:4915 (1987).Dikshit et al., Canadian J Chem, 66:2989 (1988). Froehler, et al.,Nucleic Acids Res. 16:4831 (1988). Fox, J. J., et al., J Am Chem Soc,80:1669 (1958). Gait, "Oligonucleotide Synthesis A Practical "Approach,"pp. 31-33, IRL Press (Oxford, England) (1984). Goldberg, M. L. et al;Methods in Enzymology 68:206 (1979). Greenlee, J Org Chem, 49 2632(1984). Grunstein, M. et al; Methods in Enzymology 68:379 (1979).Himmelsbach, F., and W. Pfleiderer, Tetrahedron Lett, 24:3583 (1983).Jayaraman, et al., Proc Natl Acad Sci USA 78:1537 (1981). Kamimura etal., Chem Lett (The Chem. Soc. of Japan) pg. 1051 (1983) LaPlanche etal., Nucleic Acids Res, 14: 9081 (1986). Lerman, L. S., "DNA Probes:Applications in Genetic and Infectious Disease and Cancer," Current Commin Molec Biol (Cold Spring Harbor Laboratory) (1986). Letsinger andMiller, J Amer Chem Soc, 91:3356 (1969). McBride et al., J Amer Chem Soc108:2040 (1986). Miller, et al., Biochemistry 18:5134 (1979). Miller, etal., J Biol Chem 255:6959 (1980). Miller, et al., Biochimie 67:769(1985). Murakami, et al., Biochemistry 24:4041 (1985). Niedballa, U.,and H. Vorbruggen, J Org Chem, 39:3668 (1974). Pitha, Biochem BiophysActa 204:39 (1970a). Pitha, Biopolymers 9:965 (1970b). Reese, C. B., andR. S. Saffhill, J Chem Soc PerkinTrans, 1:2937 (1972). Smith, et al.,J.A.C.S. 80:6204 (1958). Smith, et al., Proc Natl Acad Sci USA 83:2787(1986). Southern, E.; Methods in Enzymology 68:152 (1979) Stirchak E. P.et al., Organic Chem. 52:4202 (1987). Summerton, J., et al., J MolecBiol 122:145 (1978) Summerton, J., et al., J Molec Biol 78:61 (1979a).Summerton, J., J Molec Biol 78:77 (1979b). Szostak, J. W. et al; Methodsin Enzymology 68:419 (1979). Thomas, P.; Methods in Enzymology 100:255(1983). Toulme et al., Proc Nat Acad Sci USA, 83:1227 (1986).Trichtinger et al., Tetrahedron Lett 24:711 (1983).

BACKGROUND OF THE INVENTION

Polymers which are designed for base-specific binding to polynucleotideshave significant potential both for in vitro detection of specificgenetic sequences characteristic of pathogens and for in vivoinactivation of genetic sequences causing many diseases--particularlyviral diseases.

Standard ribo- and deoxyribonucleotide polymers have been widely usedboth for detection of complementary genetic sequences, and morerecently, for inactivating targeted genetic sequences. However, standardpolynucleotides suffer from a number of limitations when used forbase-specific binding to target oligonucleotides. These limitationsinclude (i) restricted passage across biological membranes, (ii)nuclease sensitivity, (ii) target binding which is sensitive to ionicconcentration, and (iv) susceptibility to cellular strand-separatingmechanisms.

In principle, the above limitations can be overcome or minimized bydesigning polynucleic acid analogs in which the bases are linked alongan uncharged backbone. Examples of uncharged nucleic acid analogs havebeen reported. Pitha et al (1970a, b) have disclosed a variety ofhomopolymeric polynucleotide analogs in which the normal sugar-phosphatebackbone of nucleic acids is replaced by a polyvinyl backbone. Thesenucleic acid analogs were reported to have the expected Watson/Crickpairing specificities with complementary polynucleotides, but withsubstantially reduced Tm values (Pitha, 1970a). One serious limitationof this approach is the inability to construct polymers by sequentialsubunit addition, for producing polymers with a desired base sequence.Thus the polymers cannot be used for base-specific binding to selectedtarget sequences.

Polynucleotide analogs containing uncharged, but stereoisomeric,methylphosphonate linkages between the deoxyribonucleoside subunits havealso been reported (Miller, 1979, 1980; Jayaraman; Murakami; Blake,1985a, 1985b; Smith). More recently a variety of analogous unchargedphosphoramidate-linked oligonucleotide analogs have also been reported(Froehler, 1988). These polymers comprise deoxynucleosides linked by the3'OH group of one subunit and the 5'OH group of another subunit via anuncharged chiral phosphorous-containing group. These compounds have beenshown to bind to and selectively block single-strand polynucleotidetarget sequences. However, uncharged phosphorous-linked polynucleotideanalogs using deoxyribonucleoside subunits are particularly costly anddifficult to prepare; the subunit starting material is quite costly andof limited availability.

More recently, deoxyribonucleotide analogs having uncharged and achiralsubunit linkages have been constructed (Stirchak 1987). These uncharged,achiral deoxyribonucleoside-derived analogs are, as mentioned above,limited by relatively high cost of starting materials.

SUMMARY OF THE INVENTION

It is one general object of the invention to provide a polymer capableof sequence-specific binding to polynucleotides and which overcomes orminimizes many of the problems and limitations associated withpolynucleotide analog polymers noted above.

The invention includes a polymer composition containing morpholino ringstructures of the form: ##STR1##

The ring structures are linked together by uncharged, chiral linkages,one to three atoms long, joining the morpholino nitrogen of one ringstructure to the 5'exocyclic carbon of an adjacent ring structure.

Each ring structure includes a purine or pyrimidine base-pairing moietyP_(i) which is effective to bind by base-specific hydrogen bonding to abase in a target sequence in a polynucleotide.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a basic β-morpholino ring structure which is linked throughuncharged, chiral linkages to form the polymer of the present invention;

FIG. 2 shows several exemplary purine and pyrimidine base-pairingmoieties (represented as Pi of the ring structures shown in FIG. 1);

FIG. 3 shows several preferred subunits suitable for forming polymershaving 5-atom (subunit A), 6-atom (subunit B), and 7-atom (subunits C-E)unit-length backbones;

FIG. 4 shows a repeating subunit segment of exemplary morpholino-basedpolymers, designated A--A through E--E, constructed using subunits A-E,respectively, of FIG. 3;

FIG. 5 shows the steps in the synthesis of several types of morpholinosubunits from a ribonucleoside;

FIG. 6 shows an alternative synthesis of the basic morpholino subunit;

FIG. 7 shows the steps in the synthesis of a morpholino subunit designedfor construction of polymers with seven-atom repeating-unit backbones;

FIG. 8 shows the binding mode for 2-amine-containing purines to polarmajor-groove sites of respective target base-pairs and a representativebase sequence of a duplex-binding polymer;

FIG. 9 shows the steps in linking two morpholino subunits through aphosphonamide linkage;

FIG. 10 shows two methods for linking morpholino subunits through aphosphoramidate linkage;

FIG. 11 shows the linking of morpholino subunits through aphosphonoester linkage;

FIG. 12 illustrates a subunit coupling procedure which concurrentlygenerates the morpholino ring structure;

FIG. 13 shows the thermal denaturation plot for a (morpholino-C)₆/poly(G) complex, where the (morpholinoC)₆ polymer was constructedaccording to the present invention; and

FIG. 14 illustrates the use of a morpholino polymer in aprobe-diagnostic system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a morpholino-based polymer which isdesigned for base-specific binding to a target sequence of apolynucleotide. The polymer is composed of morpholino-based ringstructures which are linked together by uncharged, chiral linkages, oneto three atoms long, joining the morpholino nitrogen of one structure tothe 5'exocyclic carbon of an adjacent structure.

A. Morpholino-Based Subunits

FIG. 1 shows the β-morpholino ring structures on which the polymersubunits are based, where the morpholino carbon atoms are numbered as inthe parent ribose. As seen in FIG. 1, the ring structure contains a5'methylene attached to the 4'carbon in the β-orientation.

Each ring structure includes a purine or pyrimidine or relatedhydrogen-bonding moiety, P_(i), attached to the backbone morpholinemoiety through a linkage in the β-orientation.

The purine hydrogen-bonding moieties or bases include purines as well aspurine-like planar ring structures having a 5-6 fused ring in which oneor more of the atoms, such as N3, N7, or N9 is replaced by a suitableatom, such as carbon. The pyrimidine moieties likewise includepyrimidines as well as pyrimidine-like planar 6-membered rings in whichone or more of the atoms, such as N1, is replaced by a suitable atom,such as carbon. Preferred hydrogen-bonding moieties in the inventioninclude the set of purines and pyrimidines shown in FIG. 2. Each baseincludes at least two hydrogen-bonding sites specific for apolynucleotide base or base-pair. Where the polymers are used forsequence-specific binding to single-stranded polynucleotides, the purinestructures 1-3 are designed to bind to thymine or uracil bases;structures 7-8, to guanine bases; structures 4-6, to cytosine bases; andstructure 9, to adenine bases.

The polymers of the invention are also effective to bind tohydrogen-bonding sites accessible through the major-groove in duplexpolynucleotides having mostly purine bases in one strand and mostlypyrimidine bases in the complementary strand, as discussed below.

Because of the similar type and positioning of the two central polarmajor-groove sites among the different base-pairs of duplex nucleicacids (i.e., the NH4 and O6 of a CG base-pair present the same H-bondingarray as the NH6 and O4 of an AT base-pair), the H-bonding moiety of aduplex-binding polymer must hydrogen-bond to the N7 of its targetbase-pair in order to uniquely recognize a given base-pair in a targetgenetic duplex. Thus, in the polymers of the present invention, whichare targeted against duplex genetic sequences containing predominantlypurines in one strand and predominantly pyrimidines in the other strand,the hydrogen-bonding moieties of the polymer preferably contain purineshaving an amine at the 2 position since that amine is suitablypositioned for H-bonding to the N7 of the target base-pair. Morespecifically, Structures 2 and 3 of FIG. 2 provide for specific bindingto a TA or UA base-pair while Structures 4 and 6 provide for specificbinding to a CG base-pair. Two bases which are particularly useful in aduplex-binding polymer are 2,6-diaminopurine (structure 3) and guanine(structure 4). FIG. 8A illustrates the binding of these two bases to thepolar major-groove sites of their respective target base-pairs in duplexnucleic acids. FIG. 8B illustrates a representative base sequence of apolymer designed for binding a target genetic sequence in the duplexstate.

Polymers comprising predominantly 2-amine-containing purines, thussuitable for high-specificity binding to polar major-groove sites ofduplex genetic sequences, can provide effective binding to theirtargeted genetic duplexes using alternative backbones, in addition tothe morpholino-based backbone. Examples of such alternative backbonesinclude phosphodiester-linked deoxyribonucleosides where a pendant groupon the phosphorous is one of the following: a negatively charged oxygen(i.e., the natural DNA backbone); a methyl or other alkyl group(referred to as an alkylphosphonate); a methoxy or other alkoxy group(referred to as a phosphotriester); or a mono- or dialkyl amine(referred to as a phosphoramidate).

The morpholino subunits of the instant invention are combined to formpolymers by linking the subunits through stable, chiral, unchargedlinkages. The linking group of a subunit includes aphosphorous-containing electrophile which is usually reacted with anucleophile of the subunit to which it is to be linked.

The selection of subunit linking groups for use in polymer synthesis isguided by several considerations. Initial screening of promisingintersubunit linkages (i.e., those linkages which are predicted to notbe unstable and which allow either free rotation about the linkage orwhich exist in ,a single conformation) typically involves the use ofspace-filling CPK or computer molecular models of duplex DNA or RNA. TheDNA and RNA duplexes are constructed according to parameters determinedby x-ray diffraction of oligodeoxyribonucleotides in the B-form andoligoribonucleotidecontaining duplexes in the A-form.

In each of these constructed duplexes, one of the two sugar phosphatebackbones is removed, and the prospective backbone, including themorpholino ring and intersubunit linkage, is replaced, if possible, onthe sites of the bases from which the original sugar-phosphate backbonehas been removed. Each resulting polynucleotide/polymer duplex is thenexamined for coplanarity of the Watson/Crick base pairs, torsional andangle strain in the prospective binding polymer backbone, degree ofdistortion imposed on the nucleic acid strand, and interstrand andintrastrand nonbonded interactions.

Initial studies of this type carried out in support of the inventionshow that a morpholino-based polymer has a preferred unit backbonelength (i.e., the number of atoms in a repeating backbone chain in thepolymer) of 6 atoms. However, the modeling studies also show thatcertain 5-atom and 7-atom repeating-unit morpholino-based backbones meetthe requirements for binding to targeted genetic sequences.

Since the morpholino structure itself contributes 4 atoms to eachrepeating backbone unit, the linkages in the five-atom, six-atom, andseven-atom repeating-unit backbone contributes one, two, and three atomsto the backbone length, respectively. In all cases, the linkage betweenthe ring structures is (a) uncharged, (b) chiral, (c) stable, and (d)must permit adoption of a conformation suitable for binding to thetarget polynucleotide.

Subunit backbone structures judged acceptable in the above modelingstudies are then assessed for feasibility of synthesis. The actualchemical stability of the intersubunit linkage is often assessed withmodel compounds or dimers.

FIG. 3 shows several preferred β-morpholino subunit types, includinglinkage groups, which meet the constraints and requirements outlinedabove. It will be appreciated that a polymer may contain more than onelinkage type.

Subunit A in FIG. 3 contains a 1-atom phosphorous-containing linkagewhich forms the five atom repeating-unit backbone shown at A--A in FIG.4, where the morpholino rings are linked by a 1-atom phosphonamidelinkage. It is noted here that the corresponding chiralthionyl-containing linkage (substituting an S═O moiety for thephosphorous-containing group) was found to have inadequate stability inaqueous solution.

Subunit B in FIG. 3 is designed for 6-atom repeating-unit backbones, asshown at B--B, in FIG. 4. In structure B, the atom Y linking the5'morpholino carbon to the phosphorous group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorousmay be any of the following: fluorine; an alkyl or substituted alkyl; analkoxy or substituted alkoxy; a thioalkoxy or substituted thioalkoxy;or, an unsubstituted, monosubstituted, or disubstituted nitrogen,including cyclic structures. Several cyclic disubstituted nitrogenmoieties which are suitable for the X moiety are morpholine, pyrrole,and pyrazole.

Subunits C-E in FIG. 3 are designed for 7-atom unit-length backbones asshown for C--C through E--E in FIG. 4. In Structure C, the X moiety isas in Structure B and the moiety Y may be a methylene, sulfur, orpreferably oxygen. In Structure D the X and Y moieties are as inStructure B. In Structure E, X is as in Structure B and Y is O, S, orNR.

B. Subunit Synthesis

The most economical starting materials for the synthesis ofmorpholino-subunits are generally ribonucleosides. Typically,ribonucleosides containing hydrogen-bonding moieties or bases (e.g., A,U, G, C) are synthesized to provide a complete set of subunits forpolymer synthesis. Where a suitable ribonucleoside is not available, a1-haloribose or, preferably, a 1α-bromoglucose derivative, can be linkedto a suitable base and this nucleoside analog then converted to thedesired β-morpholino structure via periodate cleavage, and closing theresultant dialdehyde on a suitable amine.

Because of the reactivity of the compounds used for subunit synthesis,activation, and/or coupling, it is generally desirable, and oftennecessary, to protect the exocyclic ring nitrogens of the bases.Selection of these protective groups is determined by (i) the relativereactivity of the nitrogen to be protected, (ii) the type of reactionsinvolved in subunit synthesis and coupling, and (iii) the stability ofthe completed polymer prior to base deprotection.

Methods for base protecting a number of the more common ribonucleosidesare given in Example 1. The methods detailed in the example aregenerally applicable for forming nucleosides with amine-protectivegroups. Standard base-protective groups used for nucleic acid chemistryare often suitable including the following groups: benzoyl for the N4 ofC; benzoyl or p-nitrobenzoyl for the N6 of adenine (A); acetyl,phenylacetyl or isobutyryl for the N2 of guanine (G); andN2,N6-bis(isobutyryl) for 2,6-diaminopurine residues. These protectivegroups can be removed after polymer assembly by treatment with ammoniumhydroxide.

It is sometimes desirable to protect the base portion of the morpholinosubunit with a group which can be readily removed by other than anucleophilic base. Suitable base protective groups removable by a strongnon-nucleophilic base via a β-elimination mechanism include:2-(4-nitrophenyl)ethoxy carbonyl or 2-(phenyl sulfonyl)ethoxycarbonylfor both the N4 of C and the N6 of A; and the 9-fluorenylmethoxycarbonyl for the N2 of G and the N2 and N6 of 2,6-diaminopurine.These groups can be removed after polymer assembly by treatment with thestrong nonnucleophilic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),under stringently anhydrous conditions.

The syntheses of representative morpholino subunits are describedparticularly in Examples 2-7. With reference to the synthesis schemedepicted in FIG. 5, a base-protected ribonucleoside is reacted withsodium periodate to form a transient 2', 3'-dialdehyde which then closesupon ammonia to form a morpholino-ring having 2' and 3'hydroxyl groups(numbered as in the parent ribose, see FIG. 1). The compound is thentreated with sodium cyanoborohydride to reduce the ring hydroxyl groups.The ring nitrogen is preferably protected by trityl derivatization or bya benzhydraloxycarbonyl group for subsequent subunit coupling. Theprotective group can be added by reacting the morpholino subunit withtrityl chloride or with nitrophenyl benzhydrl carbonate or by reactingthe dialdehyde with a primary amine, as illustrated in FIG. 6 anddescribed in Examples 3 and 5. The stereochemistry of the nucleosidestarting material is retained as long as the pH of the reaction mixtureat the iminium stage is not allowed to go above about 10.

The above synthesis results in a morpholino-ring with an available5'-hydroxyl. The 5'-hydroxyl can be converted to other active groupsincluding 5' amine and sulfhydral (Example 6) or 5'phosphonate (Example4).

In the above morpholino synthesis a variety of nitrogen sources can beused--including particularly ammonia, ammonium hydroxide, ammoniumcarbonate, and ammonium bicarbonate. Best results are obtained when thereaction solution is maintained near neutrality during the oxidation andmorpholino ring closure reactions. This can be accomplished bycontinually titrating the reaction mix or, more conveniently, by usingammonium biborate as the ammonia source. When the solution is too acidicthe yield of product is low and when it is too basic, side products(possibly due to epimerization of the 1' and/or 4' carbons) are producedwhich are difficult to separate from the desired product. It is alsonoted that the reducing agent can be added before, during, or after theoxidation step with little noticeable effect on product yield.

Ribonucleosides lacking base protection groups can also be successfullyoxidized, ring closed, and reduced in aqueous solution to generate themorpholino ring. However, without base protection the number andquantity of undesired side products frequently increases, particularlyin the case of cytidine.

The subunits formed by the above methods contain a 5'-OH, SH, or aminewhich is modified, reacted with, and/or activated, as described below,to be suitable for coupling to a second morpholino subunit. For example,FIG. 5 shows the conversion of a 5'-OH of a morpholino subunit to aphosphonyl linking moiety to form a subunit (Structure 10) which islinked to form a 5-atom unit-length backbone polymer. Details of thesubunit synthesis are given in Example 4; modification to thethiophosphonyl linking moiety is also described.

Alternatively, the subunits are designed to include aphosphorous-containing group attached directly or indirectly to themorpholino ring nitrogen, which is coupled to a 5' moiety of a secondmorpholino subunit (FIG. 7). Subunits of this type are suitable forconstructing morpholino polymers with 7-atom repeating-unit backbones.

An example of the synthesis of a subunit suitable for 7-atom unit-lengthbackbones is detailed in Example 5 (with reference to FIG. 7).

Example 7 describes, with reference to Structure E of FIG. 3, thepreparation of non-morpholino subunits which are converted intomorpholino structures during polymer assembly.

C. Activation and Coupling Reactions

The subunits prepared as above are coupled, in a controlled, sequentialmanner, often by activating the 5'hydroxyl of one subunit (having aprotected morpholino nitrogen) and contacting this activated subunitwith another subunit having an unprotected morpholino nitrogen asdescribed in Example 9. It will be recognized that different types oflinkages, such as those illustrated below, may be employed in theconstruction of a single polymer.

The simplest morpholino-type binding polymers are carbamate-linkedpolymers where the morpholino nitrogen is linked through a carbonyl tothe 5' oxygen of another subunit. Experiments conducted in support ofthe present invention demonstrate that such a polymer effectively bindsto a single-stranded DNA target sequence. However, in binding studieswith an RNA target, the polymer exhibited unusual binding, as evidencedby a highly atypical hypochromicity profile in the 320 to 230 nmspectral range and lack of a normal thermal denaturation.

Early modeling studies indicated that in a carbamate-linked polymerbound to DNA existing in a B conformation, the backbone of the polymerprovides adequate length for binding and the carbamate moieties of thepolymer backbone can assume a nearly planar conformation. This modelingresult was in good accord with the effective binding of thecarbamate-linked polmers to DNA. In contrast, similar modeling studiessuggested that binding of the carbamate-linked polymer to an RNA targetrequires one of the following: (i) the carbamate linkage of the polymeradopt a substantially nonplanar conformation, or (ii) the RNA targetsequence adopt a strained conformation in which base-stackinginteractions are quite different from that in a normal A conformation.This observation may explain the atypical binding of a carbamate-linkedpolymer to an RNA target sequence.

The modeling work further indicated that replacing the carbonylintersubunit linking moiety with either an achiral sulfonyl-containingintersubunit linkage or with a chiral phosphorous-containing linkagewould provide added length of about 0.32 angstrom per intersubunitlinkage. Such linkages would also provide greater rotational freedomabout key bonds, and bond angles of the intersubunit linkage compatiblewith an oligomer backbone conformation suitable for pairing to both RNAand DNA target sequences in their standard conformations.

The linkage in structure A--A in FIG. 4 (five-atom backbone) can beformed according to the reaction scheme shown in FIG. 9, and detailed inExample 8. Briefly, the 5'-OH of a morpholino subunit is converted to aphosphorous-containing moiety as described in Example 4. This group isactivated and coupled to a second subunit having an unprotected ringnitrogen, as shown in FIG. 9 and described in Example 8. The polymerassembly is continued by deprotecting the morpholino ring nitrogen ofthe dimer, and reacting the dimer with a third activated subunit.

The phosphoramide linkage in Structure B--B of FIG. 4 (6-atomunit-length backbone) can be formed according to the reaction schemesshown in FIG. 10 and detailed in Example 9. The 5'hydroxyl of aprotected subunit (Structure 4 of FIG. 5) is reacted with a suitablephosphorous-containing compound, such as dichloro-N,N-dimethylaminophosphate, resulting in an activated subunit. The subunit is thenreacted with a second subunit having an unprotected morpholino ringnitrogen. A large number of variations are possible in the pendant Xmoiety and, as described in Example 9, the identity of the X moietyaffects the ease of activation and coupling, the stability of theresulting linkage, and, to some extent, target-binding affinity.

In these syntheses the P═O group is essentially interchangeable with theP═S group; reactions with one are generally applicable to the other. Analternative method for forming linkages of type B--B of FIG. 4, as wellas phosphonamide and phosphonoester linkages, is to use carbodiimidecoupling: an exemplary, carbodiimide is dicyclohexylcarbodiimide (DCC).Carbodiimide coupling is described in Examples 8, 9, and 10. Byexploiting an observation of Smith et al. (1958), the carbodiimidereagent can also be used to: (a) add a phosphorous (or thiophosphorous)linking moiety to a subunit; or (b) attach a pendant X moiety to aphosphorous (or thiophosphorous) linking moiety.

Additional linkages of the type B--B (FIG. 4) can be formed byconverting the 5'hydroxyl to other functional groups (e.g., SH, CH₂, NR)before activating and coupling the subunits into polymers.

A number of 7-atom unit length backbones prepared from the morpholinosubunits (corresponding to Structures C--C and D--D in FIG. 4) alloweven more flexibility in the construction of polymers which havespecified distances between the base-pairing moieties. Using the 7-atomunit length linkages, distances between the morpholino-subunits, andconsequently between the base pairing moieties, can be lengthened. Suchlengthening of the intersubunit linkage is particularly useful whentargeting duplex genetic sequences in a B conformation.

The 7-atom backbone polymers can be readily synthesized from thesubunits C and D constructed as above, employing the general couplingreactions described in Example 10. For example, Structure C--C in FIG. 4can be produced by (a) reacting the phosphonate (or thiophosphonate)group of subunit C (FIG. 3) with a carbodiimide, and (b) coupling theactivated subunit with a second subunit having an unprotected morpholinoring nitrogen.

Similarly, structure D--D in FIG. 4 can be produced by activating thephosphonate (or thiophosphonate) with a carbodiimide, and coupling theactivated subunit with a second subunit having an unprotected 5'oxygen,sulfur, or amine, as described in Example 10.

A novel method of forming linkages corresponding to Structure D--D/E--Eof FIG. 4 entails oxidizing vicinyl hydroxyls of one subunit (FIG. 3E)and closing the resultant dialdehyde on a primary amine of anothersubunit followed by reduction with cyanoborohydride. In principle thissame scheme could also be used to couple a secondary amine of onesubunit and a mono-aldehyde of a second subunit; however, the couplingof a ribose-derived dialdehyde to a primary amine proceeds substantiallyfaster and provides a better yield. Example 7 describes the synthesis ofribonucleosides containing a primary amine at the 5'. Their use information of morpholino polymers, as illustrated in FIG. 12, isdescribed in Example 11.

D. Assembly of Polymers

After selecting a desired polymer length and recognition moiety sequence(guidelines for this are presented below), the polymer is assembledusing the general procedures described above. One method of polymerassembly involves initial preparation of an appropriate set of dimers,linking selected dimers to form tetramers, linking these to formoctamers, and so on. This method is carried out in solution,substantially according to the coupling methods described with referenceto Examples 12 and 14. Example 12 outlines such a block assemblysynthesis using monomers to form dimers, and dimers to form tetramers.It should be noted that couplings need not involve oligomers of equalsize.

A particular merit of this block assembly method is that each couplingproduct is roughly twice the length of its precursors, so purificationof the product of each coupling is simplified. Example 12 details theassembly of a 4-subunit polymer formed by this method.

The polymers may also be synthesized by stepwise subunit addition on asolid support. However, the optimal synthetic approach often uses acombination of the solution and solid support assembly methods wheredimers, trimers, or tetramers are synthesized by solution phase andsubsequently assembled into the full-length polymer on a solid support,as described in Example 13.

Typically, a solid support, such as glass beads derivatized withacid-stable, long-chain cleavable linkers, are employed as the supportmaterial, and prepared for attachment of the first subunit, or block ofsubunits, as described in Example 13. The glass beads are reacted with asubunit which generally has a readily cleavable protective group on anitrogen. Whether the morpholino subunit is linked to the support viaits morpholino nitrogen or a group at the 5' position depends on thedirection of polymer synthesis, i.e. to which group the next subunitwill be attached.

After coupling the second subunit (or oligomer which may be assembled insolution) to the support, any unreacted nucleophilic sites can be cappedby addition of a suitable capping reagent, such as p-nitrophenyl acetateor acetic anhydride, and thereafter the support is washed. Theprotecting group on the nitrogen of the terminal subunit is removed,typically by acid treatment, and after neutralization, the support isreacted with an excess of the next-in-sequence subunit (or polymer unit)which is activated by one of the methods outlined above. One feature ofthe solid support assembly method is the need for high couplingefficiencies at each subunit addition step. This high couplingefficiency is generally achieved by addition of an excess of theactivated subunit which maximizes the number of support-bound chainswhich are chain-elongated.

Chain elongation is continued in this manner, with optional capping offailure sequences after each subunit addition, until the polymer of thedesired length and sequence is achieved.

After addition of the final subunit, the terminal backbone moiety may bereacted with a suitable charged or uncharged group, as described inExample 13. The polymer is then cleaved from the support, e.g., bytreatment with either ammonium hydroxide or a non-nucleophilic basesuitable for effecting β-elimination in the linker joining the polymerto the support. The bases are deprotected and the polymer is purified asdescribed below and in Example 13.

E. Polymer Processing and Purification

Binding polymers assembled in solution (Examples 12 and 14) aretypically base-deprotected by suspending in DMSO or DMF and layering onthe suspension an equal volume of concentrated ammonium hydroxide. Thepreparation is mixed with shaking and incubated at 30° C. for 16 hrs.Workup includes removing the ammonia under reduced pressure. If aprotective group (generally trityl or a related acid-labile moiety) ispresent, this group is cleaved and the crude polymer preparation issuspended in the appropriate buffer for purification (Example 13).

Binding polymers assembled by a solid-phase method (Example 13) whereinthey are linked to the support via an ester linkage can be cleaved fromthe support by suspending the dried support in DMSO, layering on anequal volume of concentrated NH₄ OH, capping tightly, and slowlyagitating for 16 hrs at 30° C. The solid support material is removed byfiltration and the filtrate is treated as described above.

Alternatively, binding polymers linked to the support via aβ-elimination-sensitive linker can be cleaved from the support using astrong nonnucleophilic base 1,8 diazubicyclo(5.4.0.)undec-7-ene (DBU) inDMF. Using this approach one can release the polymer with its basesstill protected and thus the polymer is suitable for furthermodification and/or structural confirmation via fast atom bombardmentmass spectroscopy.

Purification of the base-deprotected polymer is preferably carried outat pH 2.5 or pH 11, depending on the pK of the base moieties in thepolymer. At pH 2.5 cytosine, adenine, and 2-6-diaminopurine moietiescarry a positive charge and guanine carries a partial positive charge.At pH 11 guanine, uracil and hypoxanthine carry a negative charge.

For polymers in which about 50% or more of the base-pairing moieties areionized at pH 2.5, the purification can be carried out by cationexchange on a column of S-Sepharose fast-flow (Pharmacia) developed witha shallow NaCl gradient buffered at pH 2.5. The effluent is monitored at254 nm and collected in a fraction collector. The full length polymer,which elutes after the shorter failure sequences, can be furtherpurified and desalted on a column of chromatographic grade polypropylene(Polysciences Inc.), eluted with an aqueous gradient of acetonitrileadjusted to pH 2.5 with formic acid, with the eluant being monitored at254 nm. The fractions containing the pure product are neutralized anddried under reduced pressure. Salts may be discarded by dissolving thepolymer in trifluoroethanol, filtering, and evaporating thetrifluoroethanol.

For polymers in which about 50% or more of the base-pairing moieties areionized at pH 11, the purification may be performed on an anion exchangecolumn of Q Sepharose fast-flow (Pharmacia) developed with an aqueous pH11 gradient of NaCl. The full-length polymer, which elutes after shorterfailure sequences, is further purified and desalted on a polypropylenecolumn eluted with an aqueous pH 11 gradient of acetonitrile. Fractionscontaining the pure product are processed as above.

The purification methods described above should be carried out so thatpolymers containing adenine base-pairing moieties are not exposed to pH11 for more than a few hours at room temperature, to avoid potentialbase lability problems. The details of the purification methods areoutlined in Example 13.

In neutral, aqueous solution, longer morpholino polymers may havesolubilities only in the sub-micromolar range. Therefore, it may beadvantageous to enhance polymer solubility by addition of one or morehydrophilic moieties, e.g., polyethylene glycol. For most of the polymertypes disclosed herein, this can be accomplished by cleaving theterminal backbone protective group from the completed polymer, andreacting the polymer, with the bases still in the protected state, withexcess of carbonyldiimidazole-activated polyethylene glycol (PEG).Thereafter the binding polymer is treated with ammonium hydroxide toremove the base-protected groups, and the polymer is purified as above.The level of solubilization is easily adjusted through proper selectionof the PEG material. Suitable PEG fractions having average molecularweights of 200, 400, 600, 1,000, 1,540, 3,400, 4,000, 6,000, 7,500, and18,500 daltons are commercially available (e.g., Polysciences, Inc.)with PEG1000 often providing the best solubilization. The solubilizingmoiety may be linked to the polymer through a cleavable linkage, ifdesired, to allow the polymer to be released from the solubilizingagent, e.g., by esterase or peptidase enzymes.

It will be appreciated that the polymer may be further derivatized orlabeled according to known procedures. For example, the polymer may beradiolabeled by preparing the polymer subunits from radiolabeledribonucleosides or by attaching a radiolabeled amino acid at oneterminus. The polymer may be readily derivatized, e.g., employingmodifications of the above subunit coupling reactions, with enzymes,chromophoric groups, or the like, where the polymer is to be used as adiagnostic probe. Further, the polymer may be derivatized withbiomolecules which serve to target the polymers to specific tissues orcell types.

F. Structural Characterization

Fully-protected binding polymers of moderate size (10 to 20 subunits)often give a strong molecular ion in FAB (Fast Atom Bombardment) massspectroscopy, providing a key confirmation of the polymer length.

Further, COSY-NMR (two-dimensional correlated spectroscopy) of thedeprotected and purified polymer provides information on the ratio ofthe different base-pairing moieties in the polymer as well asquantitative information on the ratio of binding polymer to anysolubilizing or other type moiety which may have been linked thereto.

Mobilities on ion exchange columns also provide information on thenumber of C+A base-pairing moieties in a polymer when purification iscarried out at pH 2.5 and information on the number of G+U residues whenthe purification is run at pH 11. Structural verification is easiestwhen the polymers have been assembled from oligomer blocks, such as inExamples 12, 13 and 14, since any failure sequences then differ moresubstantially from the full-length sequences.

The UV profiles of the polymers at pH 1, 7, and 13 can provideinformation about the relative nucleotide composition of the polymer.

Assessment of a morpholino-based polymer's affinity for its targetsequence is carried out by examining the melting curve of thepolymer/target duplex, as illustrated in Example 14. Further,comparisons can be made between the melting curve of a regular nucleicacid duplex (such as p(dC)₆ /p(dG)₆) and the melting curve of a hybridduplex containing a corresponding morpholino-based polymer (such as(morpholino C)₆ /p(dG)₆).

The above characterization steps have been applied to a morpholino-basedphosphordiamidate-linked poly(C) hexamer where Y is oxygen, X isN(CH₃)₂, and Z is oxygen, as described in Example 14. Characterizationof the full-length oligomer was achieved by proton NMR and negative ionFAB mass spectroscopy. With these morpholino oligomers, thefragmentation of the oligomers is greatly suppressed so that littlesequence information is available. However, the parent ion signal isquite strong and allows confirmation of the composition of themorpholino oligomer (see Example 14).

In order to increase water solubility a polyethylene glycol (PEG) tailwas attached to the oligomers. 5 equivalents of PEG 1000 was treatedwith one equivalent of bis(p-nitrophenyl)carbonate to give monoactivatedPEG. Detritylation of the hexamer with 1% acetic acid intrifluoroethanol afforded a free morpholino ring nitrogen. Treatment ofthe hexamer containing the free amine with activated PEG1000 understandard coupling conditions resulted in attachment of the PEG tail tothe hexamer. The bases were deprotected by treatment of the tailedhexamer with concentrated ammonia for 24 hours. The tailed hexamer wastaken up in pH 2.5 buffer and purified by cation exchange chromatographyon S-Sepharose Fast Flow™ eluted with a potassium chloride gradient.After neutralization the eluant was desalted on a polypropylene columneluted with a water/acetonitrile gradient. The tailed hexamer was foundto be freely soluble in pH 7.5 buffer.

The stability of complexes of the tailed hexamer with complementarynucleic acids was investigated by thermal denaturation experiments.Difference spectra between mixed and unmixed samples of the tailedhexamer and the selected phosphodiester complement were obtained from14° C. to 85° C. and over the 320 to 260 nm range (see Example 14). At60 micromolar in C monomer and 60 micromolar in G monomer the differenceUV spectrum of the tailed hexamer, (morphC)₆ with poly(dG) gave a T_(m)value of 79° C. The corresponding (morphC)₆ with poly(G) gave a Tm valueof 51.5° C. (see Example 14 and FIG. 13).

G. Diagnostic Applications

The target-specific polymers of the invention can be used in a varietyof diagnostic assays for detection of RNA or DNA having a given targetsequence. In one general application, the polymers are labeled with asuitable radiolabel or other detectable reporter group. Targetpolynucleotide, typically a single stranded polynucleotide which isbound to a solid support, is reacted with the polymer underhybridization conditions, allowed to anneal, and then the sample isexamined for the presence of polymer reporter group.

The diagnostic assay can be carried out according to standardprocedures, with suitable adjustment of the hybridization conditions toallow polymer hybridization with the target region. In this regard, itis noted that the polymer can be designed for hybridization with thetarget at a higher melting temperature than the complementarypolynucleotide strand, since polymer binding does not entail backbonecharge repulsion effects. Therefore, the polymer can bind to the targetat a temperature above the normal polynucleotide melting temperature, animportant advantage of the polymer over conventional oligonucleotideprobes. This binding at elevated temperature minimizes the problem ofcompetition for binding to the target between the probe and anycorresponding single-strand oligonucleotide which may be present in thediagnostic mixture.

In a second general type of diagnostic application, the polymers arelinked to a solid support, for capture of target RNA or DNA to thesupport. The solid support, e.g., polymeric microparticles, can beprepared by linking the polymers to the support according to the methodsdescribed above or by conventional derivatization procedures.Alternatively, where the polymers are synthesized on a solid supportthis support may also serve as the assay support.

According to an important feature of this assay system, the targetpolynucleotide molecules which are captured on the support bybase-specific binding to the polymers can be detected on the basis oftheir backbone charge, since the support-bound polymers are themselvessubstantially uncharged. To this end, the assay system may also includepolycationic reporter molecules which are designed to bind to the fullycharged analyte backbone, but not the uncharged (or substantiallyuncharged) polymer backbone, under selected binding conditions.

In one embodiment the reporter molecules are composed of a polycationicmoiety or tail designed to bind electrostatically to a fully chargedpolynucleotide, under conditions where the reporter does not bind to theless charged or uncharged binding polymer carried on the diagnosticreagent; one or more reporter groups may be attached to the tail,adapted to produce a signal by which the presence of the reporter can bedetected. Methods for forming polycationic molecules and for attachingreporter molecules to cationic compounds are known.

Each reporter molecule carries one or more reporter groups, and eachpolynucleotide can accommodate binding of typically several thousand ormore reporter molecules. Thus the system has an amplification factor, interms of reporter signal per bound analyte molecule, of several ordersof magnitude. In addition, the method has the advantage, noted above,that the polynucleotide binding reaction can be carried out underconditions in which binding competition with complementary nucleotidestrands does not occur.

The design considerations applied in preparing a polynucleotide bindingpolymer for use as a diagnostic reagent are governed by the nature ofthe target analyte and the reaction conditions under which the analyteis to be assayed. As a first consideration, there is selected anon-homopolymeric target base sequence against which the polymer isdirected. This target sequence is generally single-stranded andpreferably unique to the analyte being assayed.

The probability of occurrence of a given n-base target sequence isapproximately (1/4)^(n). Accordingly, a given n-base target sequencewould be expected to occur approximately once in a polymer containing4^(n) bases. Therefore, the probability P that a given n-base sequencewill occur in polynucleotides containing a total of N unique-sequencebases is approximately P=N/4^(n). To illustrate, the probability P thata 9-base target sequence will be found in a 20 kilobase polynucleotideis about 20×10³ /2×10⁵ or 0.08, the probability that a 16-base targetsequence will be present is about 20×10³ /4.3×10⁹ or 0.0000047. Fromthese calculations, it can be seen that a polymer having 9-16recognition moieties specific for a defined 9-16 base target sequenceshould have high specificity for the target sequence in an assay mixturecontaining only viral genomes, whose greatest complexities correspond toabout 400K of unique-sequence bases.

Similar calculations show that a 12 to 16 subunit polymer can provideadequate specificity for a viral or bacterial target sequence in anassay mixture containing viral and bacterial genomic material only;largest genomic sizes about 5,000 kilobases. A 16 to 22 subunit polymercan provide adequate specificity for a target sequence in apolynucleotide mixture containing mammalian genomic DNA material;genomic sizes of about 5 billion base pairs of unique-sequence DNA.

The polymer/analyte binding affinity, and particularly the temperatureat which the polymer just binds with the target sequence (the meltingtemperature, or Tm) can be selectively varied according to the followingcriteria: (a) number of subunits in the polymer; (b) the number ofhydrogen bonds that can be formed between the base-pairing moieties andthe corresponding, complementary bases of the analyte target sequence;(c) unit length of the polymer backbone; (d) the particular intersubunitlinkages; and (e) concentration of denaturants, such as formamide, whichreduces the temperature of melting.

From a number of studies on model nucleic acid duplexes it is known thatthe melting temperature of oligonucleotide duplexes in the 10 to 20 bprange increases roughly 3° C. per additional base pair formed by twohydrogen bonds, and about 6° C. per additional base pair formed by threehydrogen bonds. Therefore, the target sequence length originallyselected to insure high binding specificity with the polymer may beextended to achieve a desired melting temperature under selected assayconditions.

Also, where the recognition moieties used in constructing the polymerare the standard nucleic acid bases the target sequence may be selectedto have a high percentage of guanine plus cytosine bases to achieve arelatively high polymer/analyte melting temperature. On the other hand,to achieve a lower melting temperature a target sequence is selectedwhich contains a relatively high percentage of adenine plus thyminebases.

The binding components in the diagnostic system, as they function in thesolid-support diagnostic method just described, are illustrated in FIG.16. Here "S", the assay reagent, is the solid support having a number ofbinding polymers attached to its surface through spacer arms indicatedby sawtooth lines. In the assay procedure, the target DNA in singlestrand form is reacted with the support-bound polymers underhybridization conditions, and the solid support is then washed to removenon-hybridized nucleic acid material.

The washed support is then reacted with the reporter, under conditionswhich favor electrostatic binding of the reporter cationic moiety to thetarget DNA backbone. The reporter shown in FIG. 16 is a dicationicmolecule having a reporter group R.

After reaction with the reporter solution, typically at room temperaturefor 1-2 minutes, the reagent is washed to remove unbound reporter, andthen the assay reagent is assessed for bound reporter. One approach indetermining the amount of reporter associated with the reagent,particularly in the case of fluorescent or chromophoric reporter groups,is to elute the reporter from the reagent with a high salt solution andthen assess the eluate for reporter.

It is also noted here that the polymer of the invention can undergosequence-specific binding to duplex nucleic acids, viabase-pair-specific hydrogen bonding sites which are accessible throughthe major groove of the double helix. This bonding can occur in a duplexregion in which at least 70% of the bases on one strand are purines anda corresponding percent of the bases on the other strand arepyrimidines. The duplex binding polymer preferably includes2-aminopurine or 2,6-diaminopurine hydrogen bonding moieties for bindingto T-A or U-A base pairs, and guanine or thioguanine hydrogen-bondingmoieties for binding to C-G base pairs as illustrated in FIG. 8. Thus,for these special target sequences, the polymer of the invention can beused for diagnostic assays of the types just described, but where thetarget nucleic acid is in nondenatured, duplex form.

H. Other Applications

The polymers of the instant invention can be used in place of standardRNA or DNA oligomers for a number of standard laboratory procedures. Asmentioned above, morpholino-based polymers can be fixed to a solidsupport and used to isolate complementary nucleic acid sequences, forexample, purification of a specific mRNA from a poly-A fraction(Goldberg et al). The instant polymers are advantageous for suchapplications since they are inexpensive and straightforward to preparefrom activated subunits.

A large number of applications in molecular biology can be found forlabeled morpholino-based polymers. Morpholino-based polymers can beeasily and efficiently end-labelled by the inclusion in the last step ofthe polymer synthesis an activated and labelled morpholino-based subunitor, preferably, an ³⁵ S-labelled methionine, as indicated above. Thetype of label to be used is dependent on the final application of thepolymer; such labels include radioactive (³ H, ¹⁴ C, ³² P, or ³⁵ S)nucleosides or biotin. Labelled morpholino-based oligonucleotide analogscan act as efficient probes in, for example, colony hybridization(Grunstein et al), RNA hybridizations (Thomas), DNA hybridizations(Southern), and gene bank screening (Szostak et al).

The polymers of the invention also have important potential use astherapeutic agents. Recently, uncharged anti-sense nucleic acid analogs,which are nearly isostructural with DNA, have been used as anti-viraland anti-tumor agents. The polymers of the present invention provideseveral advantages over the more conventional anti-sense agents.

First, the morpholino polymers are substantially less expensive tosynthesize than oligonucleotides. This is due in part to the fact thatthe morpholino subunits used in polymer synthesis are derived fromribonucleosides, rather than the much more expensivedeoxyribonucleosides. Also, as noted above, the coupling reactionbetween a phosphorous and an amine of a second subunit occurs underrelatively mild conditions, so that protection steps and otherprecautions needed to avoid unwanted reactions are simplified. This isin contrast to standard ribo- and deoxyribonucleotide polymer synthesiswhere coupling through a phosphate ester linkage requires that thecoupling reagents be highly reactive and that the reaction be carriedout under stringent reaction/protection conditions. This advantage inpolymer synthesis also applies, of course, to diagnostic uses of thepolymer.

Second, polymer binding to its target may give substantially bettertarget inactivation, since the polymer/target duplex is not susceptibleto duplex unwinding mechanisms in the cell.

Third, the morpholino-based polymer is also more stable within the cell;the polymer backbone linkage is not susceptible to degradation bycellular nucleases.

Fourth, in therapeutic applications involving cellular uptake of thecompound, the uncharged morpholino polymer is much more likely toefficiently enter cells than a charged oligonucleotide.

In the context of therapeutic applications, the morpholino polymers ofthe present invention may be targeted against double-stranded geneticsequences in which one strand contains predominantly purines and theother strand contains predominantly pyrimidines.

Further, when a messenger RNA is coded by the mostly purine strand ofthe duplex target sequence, morpholino binding polymers targeted to theduplex have potential for also inactivating the mRNA. Thus such apolymer has the potential for inactivating key genetic sequences of apathogen in both single-stranded and double-stranded forms.

In 1981 it was reported that short (3 to 7 subunits)methylphosphonate-linked DNA analogs complementary to portions of theShine-Dalgarano consensus sequence of procaryotic mRNAs were effectivein disrupting bacterial protein synthesis in bacterial lysates and in aspecial permeable strain of bacteria. However, such agents failed toinhibit protein synthesis in normal bacteria (Jayaramon, 1981).

Experiments performed in support of the instant invention show thatpolymers of 3 to 5 subunits in length can be effective to block proteinsynthesis in normal bacteria by using a combination of bases whichresult in a high target-binding affinity. More specifically, thefollowing oligomers and oligomer combinations can perturb proteinsynthesis in normal intact bacteria (where D is 2,6-Diaminopurine orAdenine; G is Guanine; B is 5-Bromouracil, other 5-Halouracil or Uracil;and sequences are shown with their 5' end to the left): DGG, BDDG, DDGG;DGGD; GGDG; GDGG; DGGB; GGBG; GGAGG; GGDGG; and the combinationsBDD+GGDG; DDG+GDGG; DGG+DGGB; GGD+GGBG; BDDG+GDG; DDGG+DGG; DGGD+GGB;GGDG+GBG; BDD+GGDG+GBG.

The use of short binding-enhanced oligomers to disrupt the biologicalactivity of an RNA sequence which plays a key role in the metabolism ofa target class of organisms but not a correspondingly important role inhigher organisms should be broadly adaptable to a variety of pathogenicorganisms (e.g., bacteria and fungi) having a cell wall which excludesthe entrance of longer polymers.

The following examples illustrate methods of subunit and polymersynthesis, and uses of the polymer composition of the invention. Theexamples are in no way intended to limit the scope of the invention.

EXAMPLE 1 Base Protection of Ribonucleosides

The following ribonucleosides are obtained from Sigma Chemical Co. (St.Louis, Mo.): uridine, guanosine, 5-methyluridine, adenosine, cytidine,5-bromouridine, and inosine.

2,6-diamino-9-(B-D-ribofuranosyl)-9H-purine (2,6-diaminopurine riboside)is obtained from Pfaltz and Bauer, Inc., Division of Aceto Chemical Co.,Inc. (Waterbury, Conn.).

The following nucleosides are prepared by the literature methodsindicated:

1-β-D-ribofuranosyl)-2-pyrimidinone (2-hydroxy-pyrimidine riboside) isprepared by the procedure of Niedballa.

2-amino-9-β-D-ribofuranosyl)-1,6-dihydro-6hpurine-6-thione(thioguanosine) is prepared by the procedure of Fox.

Dimethoxytrityl chloride, N-6-benzoyladenosine, N-4-benzoylcytidine, andN-2-benzoylguanosine are obtained from Sigma Chemicals.9-fluorenylmethoxycarbonyl chloride (FMOC chloride),trimethylchlorosilane, isobutyric anhydride, 4-nitrobenzoyl chloride,naphthalic anhydride, and all organic solvents for reactions andchromatography were obtained from Aldrich Chemical Co. (Milwaukee,Wis.). Silica Gel is obtained from EM Science (Cherry Hill, N.J.).

When activation of the subunits is achieved using dihalogenatedelectrophiles (e.g., P(O)Cl₂ N(CH₃)₂ or P(S)Cl₂ N(CH₃)₂), better yieldsof activated subunits are often obtained by using protective groupswhich leave no acidic protons on the purine and pyrimidine exocyclicamines. Examples of such exocyclic amine moieties are as follows: the N6of adenine, the N4 of cytosine, the N2 of guanine, and the N2 and N6 ofdiaminopurine. Suitable protective groups for this purpose include thenaphthaloyl group (Dikshit) and the amidine groups developed by McBrideet al (1986). In addition, use of dihalogenated electrophiles forsubunit activation generally requires that the O6 of guanine moieties isprotected; this protection is achieved using the diphenylcarboamoylgroup (Trichtinger).

Guanosine

In order to minimize side reactions during subunit activations it isoften desirable to protect the guanine moity on both the N2 and O6 usingthe procedure of Trichtinger et al. (1983).

The N-2 9-fluorenylmethoxycarbonyl derivative of guanosine is preparedby the procedure below which is general for the protection of nucleosideamino groups: guanosine (1 mmol) is suspended in pyridine (5 ml) andtreated with trimethyl-chlorosilane (5 mmol). After the solution isstirred for 15 minutes, 9-fluorenylmethoxycarbonyl chloride (5 mmol) isadded and the solution is maintained at room temperature for 3 hours.The reaction is cooled in an ice bath and water (1 ml) is added. Afterstirring for 5 minutes conc. ammonia (1 ml) is added, and the reactionis stirred for 15 minutes. The solution is evaporated to near drynessand the residue is dissolved in chloroform (10 ml). This solution iswashed with sodium bicarbonate solution (5 ml, 10%), dried over sodiumsulfate and evaporated. The residue is coevaporated several times withtoluene and the product chromatographed on silica gel using a gradientof methanol in methylene chloride (0-50%).

N-2-Isobutyrylguanosine is prepared by the method of Letsinger. Furtherprotection of the O6 with a nitrophenethyl moiety is often desirable andcan be carried out by several methods (Gait, 1984).

N-2-acetylguanosine is obtained by the method of Reese.

N-2-naphthaylguanosine is prepared by the method of Dikshit; thisreference provides a general method for the protection of nucleosideamine groups.

Adenosine

The N-6 2-(4-nitrophenyl)-ethoxycarbonyl derivative is prepared by themethod of Himmelsbach.

N-6 (4-nitrobenzoyl)adenosine is prepared using the procedure above forFMOC-guanosine except that 4-nitrobenzoyl chloride is substituted forFMOC chloride.

The N-6 2-(phenylsulfonyl)-ethoxycarbonyl derivative is prepared by theprocedure for FMOC guanosine except the 2-(phenylsulfonyl)-ethylchloroformate (Balgobin) is used as the acylating agent andN-methylimidazole or pyridine is used as the solvent.

N-6 naphthoyladenosine is prepared by the method of Dikshit; thisreference provides a general method for the protection of nucleosideamine groups.

2,6-diaminopurineriboside

The N-2,N-6-bis(9-fluorenylmethoxycarbonyl) derivative of2,6-diaminopurine riboside is prepared by the general proceduredescribed for guanosine.

The N-2,N-6-bis(isobutyryl) derivative is prepared by the generalprocedure described for guanosine.

Thioguanosine

The N-2(9-fluorenylmethoxycarbonyl) derivative of thioguanosine isprepared by the general procedure described for guanosine.

Uridine

To minimize undesired side products during the subunit activation stepit is sometimes desirable to protect the N3 of the uracil moiety.5'O-tritylated uridine-2',3'-acetonide is converted to the N3 anisoylderivative by the procedure of Kamimura et al. (1983). The product isthen treated with hot 80% acetic acid or 0.1N HCl in THF to cleave theprotective groups on the ribose moiety.

EXAMPLE 2 Synthesis of 5'-OH Morpholino Subunits

The steps in the method are illustrated in FIG. 5, with reference tostructures shown in FIG. 5.

The base-protected ribonucleoside is oxidized with periodate to a 2'-3'dialdehyde (Structure 1). The dialdehyde is closed on ammonia or primaryamine (Structure 2) and the 2' and 3' hydroxyls (numbered as in theparent ribose) are removed by reduction with cyanoborohydride (Structure3).

An example of this general synthetic scheme is described below withreference to the synthesis of a base-protected cytosine (P_(i) *)morpholino subunit. To 1.6 l of methanol is added, with stirring, 0.1mole of N-4-benzoylcytidine and 0.105 mole sodium periodate dissolved in100 ml of water. After 5 minutes, 0.12 mole of ammonium biborate isadded, and the mixture is stirred 1 hour at room temperature, chilledand filtered. To the filtrate is added 0.12 mole sodiumcyanoborohydride. After 10 minutes, 0.20 mole of toluenesulfonic acid isadded. After another 30 minutes, another 0.20 mole of toluenesulfonicacid is added and the mixture is chilled and filtered. The solidprecipitate is washed with two 500 ml portions of water and dried undervacuum to give the tosylate salt of the free amine shown in Structure 3.

The use of a moderately strong (pKa<3) aromatic acid, such astoluenesulfonic acid or 2-naphthalenesulfonic acid, provides ease ofhandling, significantly improved yields, and a high level of productpurity.

The base-protected morpholino subunit is then protected at the annularnitrogen of the morpholino ring using trityl chloride or benzyhydralnitrophenyl carbonate (Structure 4).

Alternatively, the 5' hydroxyl can be protected with a trialkylsilylgroup.

As an example of a protection step, to 2 liters of acetonitrile isadded, with stirring, 0.1 mole of the tosylate salt from above followedby 0.26 mole of triethylamine and 0.15 mole of trityl chloride. Themixture is covered and stirred for 1 hour at room temperature afterwhich 100 ml methanol is added, followed by stirring for 15 minutes.After drying by rotovaping, 400 ml of methanol is added. After the solidis thoroughly suspended as a slurry, 5 liters of water is added, themixture is stirred for 30 minutes and filtered. The solid is washed with1 liter of water, filtered, and dried under vacuum. The solid isresuspended in 500 ml of dichloromethane, filtered, and rotovaped untilprecipitation just begins, after which 1 liter of hexane is added andstirred for 15 minutes. The solid is removed by filtering, and driedunder vacuum.

The above procedure yields the base-protected morpholino subunittritylated on the morpholino nitrogen and having a free 5' hydroxyl(Structure 4).

EXAMPLE 3 Alternative Synthesis of Morpholino Subunits

This example describes an alternative preparation of a morpholinosubunit containing an acid-labile moiety linked to the morpholino ringnitrogen. The steps are described with respect to FIG. 6.

The subunit is prepared by oxidizing a ribonucleoside with periodate, asin Example 2, and closing the resultant dialdehyde (Structure 1) on theprimary amine 4,4'-dimethoxybenzhydrylamine (which can be prepared bythe method of Greenlee, 1984) buffered with benzotriazole, orp-nitrophenol. Reduction with sodium cyanoborohydride, carried out as inExample 2, gives a morpholino subunit (Structure 2) having a4,4,-dimethoxybenzhydryl group on the morpholino nitrogen.

It is noteworthy that this procedure is particularly useful forpreparing morpholino subunits from ribonucleosides which do not have aprotective group on the base (e.g., uridine).

EXAMPLE 4 Synthesis of 5'-phosphonate Subunit

The steps in the method are described with reference to structures inFIG. 5.

The 5' hydroxyl of the doubly-protected morpholino subunit (Structure 4)is converted to a phosphonate as follows.N4-Benzoylcytidine-2',3'-acetonide (1 mmole) is converted to the 5'-Iododerivative by reaction with methyltriphenoxyphosphonium iodide in DMF(20 ml) under argon at room temperature for 20 hours. Methanol (5 ml) isadded to the reaction mixture and after 30 minutes the mixture isevaporated in vacuo. The residue is dissolved in ethyl acetate and thesolution washed first with aqueous sodium thiosulfate and then withbrine. After drying with sodium sulfate and evaporation of the solvent,the product is purified by chromatography on silica usingisopropanol/chloroform solvents.

The iodide derivative prepared above is treated with a large excess ofanhydrous phosphine in ethanol for two days at 50° C. is a well-sealedvessel. At the end of this time the vessel is cooled, vented, and theexcess phosphine allowed to slowly evaporate. The expelled vapors arebubbled into a solution containing sodium hypochlorite to decompose thetoxic gas. The alcoholic solution of the primary phosphine is treated,while cooling the solution, with solid sodium carbonate and an excess of30% hydrogen perioxide with cooling. The product phosphonic acid ispurified by ion exchange chromatography on an anion exchange column.

This dianionic phosphate product, where the counter ions aretriethylammonium, is mixed with an excess of reagent which wll result inthe carbodiimide-mediated addition of the desired pendant X moiety (FIG.3a). Examples of suitable reagents are as follows: addition of methanolgives X=methoxy; and addition of dimethylamine gives X=N(CH₃)₂. To thismixture a carbodiimide, such as DCC, is added. The resulting subunit isof the form shown in FIG. 3a. A substantially less basic counter ion(e.g., pyridine) should not be used, since it would allow two X moietiesto be added to the phosphonate moiety.

EXAMPLE 5 Synthesis of N-methanephosphonate Morpholino Subunit

This example describes the preparation of a subunit containing amethylphosphonate moiety linked to the morpholino ring nitrogen suitablefor preparing polymers (FIG. 3d) with 7-atom unit-length backbones. Thesteps are described with respect to structures shown in FIG. 7.

A base-protected ribonucleoside is reacted with di(p-methoxy)tritylchloride to give Structure 1. The ribose moiety is then oxidized withperiodate in the presence of aminomethanephosphonic acid (AMPA) andN-ethyl morpholine. The oxidation is followed by reduction with sodiumcyanoborohydride in the presence of benzotriazole (used to buffer thereaction mix) to give a morpholino subunit having a methanephosphonicacid group on the morpholino nitrogen (Structure 2). Thereafter theproduct is purified by silica gel chromatography developed with achloroform/methanol mixture 1% in triethylamine.

This dianionic phosphonate product, where the counter ions aretriethylammonium, is mixed with an excess of reagent which will resultin the addition of the desired pendant X moiety (FIG. 3d). Examples ofsuitable reagents are as follows: addition of methanol gives X=methoxy;and addition of dimethylamine gives X=N(CH₃)₂. To this mixture acarbodiimide, such as dicyclohexylcarbodiimide (DCC) is added to giveStructure 3 of FIG. 7. If a substantially less basic counter ion (e.g.,pyridine) is used, then two X moieties may add to the phosphonatemoiety.

EXAMPLE 6 Conversion of 5'Hydroxyl to 5'Amine and to 5'Sulfhydral

The steps in the synthesis are described with reference to structuresshown in FIG. 5.

A. Conversion to the Amine

The 5'hydroxyl of the doubly-protected morpholino subunit (Structure 4,FIG. 5) can be converted to an amine as follows. To 500 ml of DMSO isadded 1.0 mole of pyridine (Pyr), 0.5 mole of triflouroacetic acid(TFA), and 0.1 mole of the morpholino subunit. The mixture is stirreduntil all components are dissolved and then 0.5 mole of eitherdiisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC) isadded. After 2 hours the reaction mixture is added to 8 liters ofrapidly stirred brine. This solution is stirred for 30 minutes and thenfiltered. The resultant solid is dried briefly, washed with 1 liter ofice cold hexanes and filtered. The solid is added to 0.2 mole of sodiumcyanoborohydride in 1 liter of methanol and stirred for 10 minutes. Tothis mixture, 0.4 mole of benzotriazole or p-nitrophenol is added,followed the addition of 0.2 mole of methylamine (40% in H₂ O). Thepreparation is stirred four hours at room temperature. [Note: thebenzotriazole or p-nitrophenol buffers the reaction mixture to preventracemization at the 4' carbon of the subunit at the iminium stage of thereductive alkylation.] Finally, the reaction mixture is poured into 5liters of water, stirred until a good precipitate forms, and the solid(Structure 6, FIG. 5) is collected and dried.

B. Conversion to the Sulfhydral

The 5' hydroxyl of the doubly-protected morpholino subunit is convertedto a sulfhydral as follows. One-tenth mole of the 5'-hydroxyl subunit(Structure 4, FIG. 5) is added to 1 liter of pyridine, followed by theaddition of 0.12 mole of toluenesulfonylchloride. This solution isstirred for 3 hours at room temperature and the reaction yeilds theproduct shown as Structure 7 of FIG. 5. The pyridine is removed byrotovapping, and to the solid is added 0.5 mole of fresh sodiumhydrosulfide in 1 liter of methanol. This reaction mixture is stirred atroom temperature overnight. The mixture is added to 5 liters of water,stirred 20 minutes, and the resulting solid material is collected byfiltration and dried to give the product shown as Structure 8 of FIG. 5.

EXAMPLE 7 Synthesis of 5'Aminomethylphosphonate Riboside Subunit

This example describes the preparation of a riboside subunit containingan aminomethylphosphonate moiety linked to the riboside. The structuresreferred to in this example are shown in FIG. 12.

Aminomethylphosphonic acid (Aldrich Chem. Co.) is reacted with tritylchloride in the presence of triethylamine. The dianionic phosphonateproduct, where the counter ions are triethylammonium, is mixed with anexcess of reagent suitable for adding the desired pendant X moiety(e.g., addition of methanol gives X=methoxy, and addition ofdimethylamine gives X=N(CH₃)₂) and then a carbodiimide, such asdicyclohexylcarboiimide (DCC), is added. The resultant monoanionicproduct is shaken with a mixture of water and chloroform containingpyridinium hydrochloride. This procedure gives a monoionic phosphonicacid having a pyridinium counter ion. This product is added tochloroform containing N4-Benzoylcytidine-2',3'-phenylboronate and DCC isadded. The product is dried and chromatographed on silica usingmethanol/chloroform mixtures. The pure product is next treated with1,3-dihydroxypropane to give Structure 2 of FIG. 12, and a portion isfurther treated with acetic acid in trifluoroethanol to give Structure1.

EXAMPLE 8 Coupling to Give Phosphonamide Linkage

This example describes the coupling of a phosphonate subunit, preparedas in Example 4, with a second subunit having a free morpholino ringnitrogen. The Example is descried with reference to structures in FIG.9.

The starting material is the base-protected, morpholino nitrogenprotected phosphonate subunit prepared in Example 4. A triethylaminesalt of this subunit is suspended in chloroform and shaked with watercontaining pyridinium hydrochloride, resulting in the pyridine salt(Structure 1) of the subunit in the chloroform phase. The organic phaseis separated, washed with water, and dried. One portion of the resultingsolid is mixed with an escess of 3-hydroxypropionitrile; DCC is thenadded to the mixture. The product of the resulting reaction isdetritylated with 2% acetic acid in trifluoroethanol to give Structure2. Structures 1 and 2 are then mixed in the presence of DCC resulting inthe coupled dimer shown as Structure 3. This dimer may be selectivelydeprotected on either end for assembly into longer oligomer blocks orpolymers. The cyanoethyl group may be removed with DBU in a nonproticsolvent (e.g., DMF) or the trityl moiety may be cleaved as describedabove.

EXAMPLE 9 Activation and Coupling to Give Phosphoramide Linkages A.X=--CH₃

Example 9A describes the coupling of a 5'hydroxyl subunit, prepared asin Example 2 or 3, to a second subunit having a free morpholino ringnitrogen to give an alkylphosphonamidate intersubunit linkage. Theexample is described with reference to structures in FIG. 10 where X isan alkyl group.

One mmole of 5'hydroxyl subunit, base-protected and tritylated on themorpholino nitrogen (Structure 4 of FIG. 5), is dissolved in 20 ml ofdichloromethane. To this solution 4 mmole of N-ethylmorpholine and 1.1mmole of methylphosphonic dichloride, for Z═O (or methylthiophosphonicdichloride, for Z═S), are added, followed by the addition of 1 mmole ofN-methylimidazole. After one hour the reaction solution is washed withaqueous NaH₂ PO₄. The activated subunit is isolated by chromatography onsilica gel developed with ethyl acetate (Structure 2 of FIG. 10 where Xis methyl). This activated subunit can be directly linked to themorpholino nitrogen of a second subunit (Structure 3) by mixing them inDMF. This coupling reaction yields the dimer shown as Structure 4 (whereX is --CH₃).

Alternatively, a small amount of water can be added to the reactionsolution instead of washing with NaH₂ PO₄. The resulting solution isstirred for 10 minutes to effect conversion of the activated subunit(Structure 2) to the phosphonate salt (Structure 5, where X is methyland the counter ion is N-ethylmorpholine). The product is purified bysilica gel chromatography developed with a methanol/chloroform mixture1% in triethylamine. The purified product is shaken withchloroform/water containing 2% pyridinium hydrochloride to change thecounter ion to pyridinium. After drying, the product is suitable forcoupling to a 5'-protected subunit, having a free morpholino nitrogen(Structure 6), using DCC (dicyclohexylcarbodiimide); preferably, thereaction is carried out in dichloromethane. The carbodiimide couplingreaction yields the dimer shown as Structure 7 (where X is methyl).

The alkylphosphonoamidate intersubunit linkage is very stable to ammoniaused for base deprotections. In contrast, the linkage is sensitive tostrong acids. For instance, the linkage has a half time of cleavage ofabout 3 hours in 2% dichloroacetic acid in dichloromethane. However, thelinkage showed no detectable cleavage after 18 hours in 2% acetic acidin trifluoroethanol, conditions suitable for detritylation of themorpholino nitrogen.

B. X=--O--CH₂ CH₃

Example 9B describes the coupling of a 5'hydroxyl subunit, prepared asin Example 2 or 3, to a second subunit having a free morpholino ringnitrogen to give a phosphodiesteramide intersubunit linkage. The exampleis described with reference to structures in FIG. 10 where X is analkoxide group.

One mmole of 5'hydroxyl subunit, base-protected and tritylated on themorpholino nitrogen (Structure 4 of FIG. 5), is suspended in 80 ml ofbenzene and 2.2 mmole of N-methylimidazole is added. After the subunitis dissolved 1.2 mmole of ethyl dichlorophosphate for Z═O (orethyldichlorothiophosphate for Z═S) are added. After an hour thereaction solution is washed with aqueous NaH₂ PO₄. The activated subunitis isolated by chromatography on silica gel developed with ethyl acetate(Structure 2 in FIG. 10, where X is --O--CH₂ CH₃). This activatedsubunit can be directly linked to the morpholino nitrogen of a secondsubunit (Structure 3) by mixing in DMF. This coupling reaction yieldsthe dimer shown as Structure 4.

When ethyldichlorothiophosphate (Z═S) is used for activation of thesubunits, improved yields are obtained with the following modifications.One mmole of 5' hydroxyl subunit, base-protected and tritylated on themorpholino nitrogen (Structure 4 of FIG. 5), is suspended in 20 ml ofchloroform. To this solution 1 ml of N-methylimidazole is added,followed by the addition of 1.6 ml of ethyldichlorothiophosphate(Aldrich Chem. Co.). After 1 hour the subunit product is purified bysilica gel chromatography developed with 20% acetone/80% chloroform.This activated subunit (Structure 2, where X is --O--CH₂ -CH₃ and Z issulfur) can be coupled to the morpholino nitrogen of a second subunit asdescribed above.

Alternatively, a small amount of water can be added to the reactionsolution instead of washing with NaH₂ PO₄. The resulting solution isstirred for 10 minutes to effect conversion of the activated subunit(Structure 2) to the phosphonate salt (Structure 5, where X is --O--CH₂CH₃ and the counter ion is a protonated N-methylimidazole). The productis purified by silica gel chromatography developed with amethanol/chloroform mixture 1% in triethylamine. The purified product isshaken with chloroform/water containing 2% pyridinium hydrochloride tochange the counter ion to pyridinium. After drying the product issuitable for coupling to a 5'-protected subunit having a free morpholinonitrogen (Structure 6) using DCC (dicyclohexylcarbodiimide) indichloromethane. The carbodiimide coupling reaction yields the dimershown as Structure 7 (where X is --O--CH₂ CH₃).

C. X=--F

Example 9C describes the coupling of a 5'hydroxyl subunit, prepared asin Example 2 or 3, to a second subunit having a free morpholino ringnitrogen to give a fluorophosphoramidate intersubunit linkage. Theexample is described with reference to structures in FIG. 10 where X isa fluorine.

The starting material is one mmole of 5'hydroxyl subunit, base-protectedwith groups removable by a beta elimination mechanism and tritylated onthe morpholino nitrogen (Structure 4 of FIG. 5). The subunit isdissolved in 20 ml of dichloromethane to which is added 6 mmole ofN-methylimidazole, followed by the addition of 2.5 mmole offluorophosphoric acid. 5 mmole of DCC is added and the solution stirredthree hours. The reaction solution is washed with aqueous NaH₂ PO₄ andthe organic phase dried under reduced pressure to give thefluorophosphate salt (Structure 5 where X is F and the counter ion isN-methylimidazole). The product is purified by silica gel chromatographydeveloped with a methanol/chloroform mixture 1% in pyridine to give thepyridinium salt. After drying the purified product is suitable forcoupling to a 5'-protected subunit, having a free morpholino nitrogen(Structure 6), using DCC in dichloromethane. This carbodiimide couplingreaction yields the dimer shown as Structure 7 (where X is F).

Polymers containing the fluorophosphoramidate intersubunit linkageshould not be exposed to strong nucleophiles, such as ammonia.Consequently, bases of the subunits used for assembling such polymersshould be protected with groups which can be cleaved without the use ofstrong nucleophiles. Protective groups cleavable via a beta eliminationmechanism, as described in Example 1, are suitable for this purpose.

D. X=N(CH₃)₂

Example 9D describes the coupling of a 5'hydroxyl subunit, prepared asin Example 2 or 3, to a second subunit having a free morpholino ringnitrogen to give a phosphordiamidate intersubunit linkage. The exampleis described with reference to structures in FIG. 10, where X is adisubstituted nitrogen.

One mmole of 5'hydroxyl subunit, base-protected and tritylated on themorpholino nitrogen (Structure 4 of FIG. 5) is dissolved in 5 ml ofdichloromethane. Six mmole of N-ethylmorpholine and 2 mmole ofdimethylaminodichlorophosphate (OP(Cl)₂ N(CH₃)₂) for Z═O (or thethiophosphate analog for Z═S) is added to the solution, followed by theaddition of 0.5 mmole of N-methylimidazole. After the reaction iscomplete (assessed by thin layer chromatography) the reaction solutionis washed with aqueous NaH₂ PO₄. The activated subunit is isolated bychromatography on silica gel developed with acetone/chloroform(Structure 2 in FIG. 10, where X is N(CH₃)₂). The activated subunit isdirectly linked to the morpholino nitrogen of a subunit (Structure 3) inDMF containing triehtylamine sufficient to neutralize the HCl producedin the reaction, to give the dimer shown as Structure 4.

The dimethylaminodichlorophosphate (X is --N(CH₃)₂ and Z is oxygen) usedin the above procedure was prepared as follows: a suspension containing0.1 mole of dimethylamine hydrochloride in 0.2 mole of phosphorousoxychloride was refluxed for 12 hours and then distilled (boiling pointis 36° C. at 0.5 mm Hg). The dimethylaminodichlorothiophosphate (X is--N(CH₃)₂ and Z is sulfur) used above was prepared as follows: asuspension containing 0.1 mole of dimethylamine hydrochloride in 0.2mole of thiophosphoryl chloride was refluxed for 18 hours and thendistilled (boiling point 85° C. at 15 mm Hg).

EXAMPLE 10 Coupling to Give Phosphonester Linkage

This example describes the coupling of a methylphosphonate subunit,prepared as in Example 5, with a second subunit having a free5'hydroxyl. The example is described with reference to structures inFIG. 11.

The starting material is the base-protected, morpholinonitrogen-protected methyl phosphonate subunit prepared as in Example 5.A triethylamine salt of this subunit is suspended in chloroform andshaken with water containing 2% pyridinium hydrochloride, resulting inthe pyridinium salt (Structure 1) of the subunit in the chloroformphase. The organic phase is separated, washed with water, and dried. Oneportion of the resulting solid is mixed with an excess of3-hydroxypropionitrile; and 2 equivalents of DCC is then added to themixture. The product of the resulting reaction is detritylated with 1%dichloroacetic acid in dichloromethane to give Structure 2. Structures 1and 2 are then mixed in the presence of DCC resulting in the coupleddimer shown as Structure 3. This dimer may be selectively deprotected oneither end for assembly into longer oligomer blocks or polymers; thecyanoethyl group may be removed with DBU in a non-protic solvent (e.g.,DMF), or the dimethoxytrityl moiety may be cleaved as above.

EXAMPLE 11 Simultaneous Morpholino Ring Formation and Subunit Coupling

This example describes the oxidation of a ribonucleoside containing aprotected amine linked through the 5' methylene, such as prepared inExample 7, and coupling to the unprotected amine of another subunit tosimultaneously form a morpholino ring structure and join the subunits.The example is described with reference to the structures in FIG. 12.

Amine Protection

Ten mmole of ribonucleoside containing a 1° amine linked through the 5'methylene (Structure 1) is reacted with 11 mmole of trityl chloride toprotect the amine (Structure 2).

Oxidation

The tritylated subunit (Structure 2), in methanol, is reacted with 11mmole of NaIO₄ to give the dialdehyde (Structure 3).

Coupling

If the coupling solution is too acidic the reaction is very slow and ifthe solution is too basic, epimerization of the Structure 3 componentappears to occur. A weak acid is used to neutralize the amine component(Structure 1) and buffer the reaction in this coupling step. Weak acidswhich have been found suitable for this purpose are: carbonic, ortho andpara nitrophenol, and benzotriazole. Accordingly, the dialdehyde(Structure 3) is combined with a suitable salt of Structure 1 in awater/methanol mixture to give the coupled product (Structure 4).

Reduction

Either during or after the morpholino ring closure step sodiumcyanoborohydride is added to reduce the dihydroxymorpholino ring(Structure 4) to the desired morpholino product (Structure 5).

EXAMPLE 12 Solution-Phase Block Assembly of Phosphordiamidate-LinkedOligomer of the Sequence 5'-CUGU

This example describes the assembly of a short oligomer containing aphosphordiamidate-linked backbone (Structure B--B of FIG. 4, where X isN(CH₃)₂, Y is oxygen and Z is oxygen) coupled as in Example 9. Thissolution assembly method is particularly useful for large-scalesynthesis of short oligomers suitable for subsequent assembly intolonger oligomers using the solid-phase method (Example 13).

5'OH morpholino subunits of C, U, and G tritylated on the morpholinoring nitrogen are prepared as in Example 2 or 3. The U subunit is thenactivated by conversion to the monochlorophosphoramidate form as inExample 9D. The C subunit and the G subunit are deprotected withtrifluoroethanol (TFE) containing 2% acetic acid; the TFE is thenremoved under reduced pressure. The residue is washed with ether toremove any residual acetic acid. The deprotected C component (1.1 mmole) is dissolved in 5 ml DMF and 0.3 ml TEA, followed by addition of1.0 m mole of the activated U component. Likewise, the deprotected Gcomponent is reacted with the activated U component.

After one hour each of these preparations is added to 100 ml of rapidlystirred brine and the solid collected and washed with water. The GUdimer is dried thoroughly under high vacuum and then activated as inExample 9D. The best tetramer coupling results are obtained whenpurification of the dimer, via silica gel chromatography, is carried outafter, rather than before, this activation step.

The CU dimer is deprotected as above. Better yields of tetramer areobtained when the dimer, after the initial ether precipitation, isthoroughly resuspended in about 2 ml of trifluoroethanol, reprecipitatedwith 30 ml of ether, and then resuspended in DMF and TEA for subsequentcoupling.

Coupling to form the desired tetramer entails simply adding 1 m mole ofactivated GU dimer to the DMF/TEA solution containing 1.1 mmole ofdeprotected CU dimer.

Workup of the tetramer entails adding the reaction mixture to brine,washing the solid with water, and drying under vacuum to give thedesired tetramer: 5'-CUGU having a hydroxyl at the 5' end and a tritylon the morpholino nitrogen of the U subunit. The structure of thistetramer is most easily confirmed by negative ion fast atom bombardmentmass spectroscopy. As a rule the dominant specie in such spectra is themolecular ion.

EXAMPLE 13 Solid-Phase Assembly of Sulfamide-Linked Morpholino Polymer

This example describes the use of tetramer blocks, prepared as inExample 12, for solid-phase assembly of a morpholino polymer containingphosphordiamidate intersubunit linkages. Solid-phase assembly provides arapid method for assembly of longer binding polymers. The use of shortoligomer blocks instead of monomers greatly simplifies separation of thefinal product from failure sequences.

A. Synthesis of short oligomers

The following tetramers are synthesized in solution: 5'-CUGU (Example12); 5'-UCGG; 5'-GCGC; 5'-CACU. These tetramers are converted to theiractivated monochloro form by the general method described in Example 9D.

B. Preparation of the first monomer with a cleavable linker andattachment to the solid support

Morpholino C subunit containing a trityl moiety on the morpholino ringnitrogen and having a methylamine on the 5' methylene, prepared as inExample 6, is reacted with a 3-fold molar excess ofBis[2-(succinimidooxycarbonyloxy)ethyl]sulfone from Pierce of Rockford,Ill., USA. This product is purified by silica gel chromatography andthen added to a suitable solid support containing primary aminefunctions (e.g., Long Chain Alkyl Amine Controlled Pore Glass, fromPierce of Rockford, Ill.). This procedure links the first tritylatedsubunit to the synthesis support via a linker which is stable to theacidic conditions used for detritylations, but which can be readilycleaved via a beta elimination mechanism using a strong non-nucleophilicbase, such as a 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

C. Stepwise assembly of the polymer bound to the solid support

The coupling cycle for addition of each subunit or oligomer blockgenerally includes deprotection of the terminal backbone moiety, athorough wash, addition of the next activated subunit or oligomer block,and a thorough wash. The coupling efficiency for each addition can bedetermined by collecting each detritylation solution and subsequent washand quantitating the trityl therein.

Detritylation in the present polymer is achieved by slowly passingthrough the column a solution of 2% acetic acid in trifluoroethanoluntil the eluant no longer tests positive for trityl (readily determinedby adding a drop of eluant to 100 μl methanesulfonic acid and inspectingfor the visible yellow color characteristic of the trityl carboniumion). Thereafter the support is thoroughly washed to remove excess acidand then washed with DMF containing 1% by volume of N-ethylmorpholine(NEM). Coupling of the next subunit or oligomer block in the desiredpolymer sequence entails addition of a concentrated DMF solutioncontaining the activated monomer or oligomer and a molar equivalent ofNEM. Since the rate of coupling is a function of concentration it isdesirable to add a substantial molar excess of monomer or oligomerrelative to the concentration of support-bound growing chains. A 5-foldmolar excess of activated monomer or oligomer over that of the growingchains generally gives acceptable coupling efficiencies. Requiredcoupling times can be determined by removing at specified time intervalssmall defined quantities of the support material, thoroughly washing,treating the support with methanesulfonic acid, and thenspectrophotometrically quantitating the released trityl carbonium ion(molar absorbance at 409 nm is 45,000 in methanesulfonic acid). Aftercoupling is complete the unreacted subunit or oligomer is washed fromthe support with DMF. The unreacted subunit is generally recovered,purified by chromatography, and reused for later synthesis. The supportis thoroughly washed with the solvent trifluoroethanol, without addedacid. Washing is complete when addition of a drop of the wash eluant to100 μl methanesulfonic acid shows no yellow color.

The above coupling cycle is used to add, in order, the four activatedtetramers 5'-CUGU, 5'-UCGG, 5'-GCGC, and 5'-CACU. This results in thefollowing polymer: support-linker-CCUGUUCGGGCGCCACU-trityl.

D. Cleavage from the support

The synthesis support is treated with 20% DBU in DMF for two hours atroom temprature in the presence of 2% diethylmalonate, to tie up thevinylsulfone generated during cleavage of the linker. The releasedmorpholino polymer is washed from the support with DMF and precipitatedby adding ethylacetate. The precipitate contains full-length polymerhaving a 5' methylamine, the bases still protected and a trityl moietyon the terminal morpholino nitrogen. In addition, the precipitatecontains small amounts of failure sequences. At this stage the polymersize can be confirmed by positive ion fast atom mass spectrometry.

E. Addition of solubilizing moieties

If it is desired to add two solubilizing groups to the morpholinopolymer this can be done conveniently by detritylting the N-terminalmorpholino nitrogen using 2% acetic acid in trifluoroethanol.Alternatively, if only one solubilizing moiety is to be added, then the5'methylamine is acetylated with acetic anhydride before thedetritylation step.

Polyethylene glycol 1000 (from Polysciences Inc., Warrington, Pa., USA)is thoroughly dried by dissolving in dry DMF and then evaporating thesolvent under vacuum. The solid is resuspended in a minimal volume ofpure dry DMF and 0.5 mole equivalent (relative to PEG 1000) ofbis(p-nitrophenyl)carbonate and 1 mole equivalent of TEA is added andthe preparation sealed and incubated overnight at 30° C. to givep-nitrophenyl carbonate-activated PEG 1000.

The full-length morpholino polymer which has been detritylated is addedto a substantial molar excess (generally 10- to 20-fold) of activatedPEG 1000 and incubated two hours at room temperature. Unreacted PEG 1000is removed by precipitation of the tailed polymer with ether. The tailedpolymer is then air-dried.

F. Base deprotection

The dried polymer is suspended in DMSO, the DMSO solution chilled, andan equal volume of concentrated NH₄ OH is carefully layered on top ofthe chilled DMSO, and the container tightly capped. The preparation isincubated at 30° C. for eighteen hours. Thereafter, the solution isbriefly exposed to aspirator vacuum to remove ammonia.

G. Purification of morpholino polymer

Purification at pH 2.5 is generally used for binding polymers whereabout half or more of the base-pairing moieties are of types 1, 2, 3,and 7 of FIG. 2.

Water to be used for chromatography is degassed under aspirator vacuumand phosphoric acid added to give pH 2.5 (solvent A). A corresponding pH2.5 solution is made 2N in KCl (solvent B). Solvent A is mixed 1:1 byvolume with chromatographic-grade acetonitrile to give solvent C.

Load up to about 10 mg of the polymer in 10 ml of solvent A on achromatography column 1 cm in diameter and 10 to 20 cm in length whichis packed with the cation-exchange support S-Sepharose Fast Flow(Pharmacia). Proportionately larger quantities can be loaded on largercolumns of the same length, e.g., up to 60 mg can be loaded on a 2.5 cmdiameter column and 250 mg on a 5 cm diameter column. After washing thecolumn thoroughly with solvent A elute with a linear gradient rangingfrom 100% solvent A to 100% solvent B and monitor the eluant to 254 nm.The desired binding polymer is generally the last and the largest peakto elute from the column. When the polymer is prepared by blockassembly, base-line separations are often achieved. When peak shapes areunsymmetrical the problem generally has been due to insolubility of thebinding polymer rather than a lack of capacity of the chromatographicpacking. Such a problem, which is most common when the binding polymersdo not contain a PEG solubilizing tail, can often be solved by reducingthe quantity of binding polymer loaded in a given run. When peaks aresymmetrical but base-line separation is not achieved, substantialimprovements are usually attained simply by eluting with a shallowergradient.

The eluant containing the polymer is desalted by loading on anequivalent-sized column packed with 35 micron chromatographicpolypropylene (cat. no. 4342 from Polysciences, Inc.) and washingthoroughly with solvent A. If baseline separation was achieved in theforegoing cation exchange chromatography, then pure product is obtainedsimply by eluting with solvent C; otherwise, the product is eluted witha linear gradient ranging from 100% solvent A to 100% solvent C. Whenthe product is somewhat acid sensitive the eluant is neutralized withdilute NaOH before drying under reduced pressure.

Purification at high pH

Purification at pH 11 is generally used for binding polymers where abouthalf or more of the base-pairing moieties are of types 4, 5, 6 and 9 ofFIG. 2.

N,N-diethylethanolamine (Aldrich) is added to degassed water to adjustthe pH to 11.0 (solvent D). A corresponding pH 11 solution 2N in KCl(solvent E) is prepared. A third pH 11 solution is prepared by mixingSolvent D 1:1 by volume with chromatographic grade acetonitrile (solventF).

The fully-protected binding polymer, prepared as above, is suspended insolvent D at a concentration of about 100 μg/ml. The pH is adjusted, ifnecessary, to pH 11 with N,N-diethylethanolamine. About 10 ml of thispolymer solution is placed on a chromatography column 1 cm in diameterand 10 to 20 cm in length which is packed with anion-exchange supportQ-Sepharose Fast Flow (Pharmacia). After washing the column thoroughlywith solvent D, the column is eluted with a linear gradient ranging from100% solvent D to 100% solvent E and the eluant is monitored at 254 nm.

The eluant containing the polymer is desalted by loading on anequivalent-sized column of polypropylene and washing thoroughly withsolvent D. If baseline separation is achieved in the foregoing anionexchange chromatography then pure product is obtained simply by elutingwith solvent F; otherwise, the product is eluted with a linear gradientranging from 100% solvent D to 100% solvent F. Fractions containing theproduct are dried under reduced pressure.

H. Sequence confirmation

While mass spectral analysis of the full-length polymer in thefully-protected state, as described earlier, does serve to confirm boththe polymer length and the base composition, it does not provideinformation on the subunit sequence. Significant sequence informationcan be obtained from fragmentation patterns of deoxyribonucleic acidsand carbamate-linked deoyribonucleoside-derived polymers (Griffin etal., (1987), Biomed. & Environ. Mass Spectrometry 17 105), however, manyof the morpholino polymers of the instant invention are quite resistantto fragmentation and give predominantly the molecular ion with onlyminimal fragments.

One method for confirming the sequence of the polymer is to take a smallportion of the growing polymer after coupling each oligomer block anduse mass spectral analysis to follow the elongation of the polymer. Thismethod is applicable except for those rare cases where two blocks usedin the synthesis happen to have exactly the same mass.

An indirect method to help verify the correctness of the polymer subunitsequence is to pair the morpholino polymer with its complementary DNA(whose sequence can be confirmed by established methods) and with DNAsequences which might have resulted if the blocks were assembled in thewrong order. Pairing between the polymer and DNA can be evaluated bylooking to a hypochromic shift in the 240 to 290 nm wavelength region.Such a shift occurs only between the polymer and its complementarysequence. The polymer/DNA duplex can also be distinguished from anypartially-mismatched duplex by slowly raising the temperature whilemonitoring the absorbance in the 240 nm to 290 nm region. The perfectduplex will have a melting temperature (corresponding to a 50% reductionin the hypochromicity) generally 10 degrees or more above that of anymismatched duplex.

EXAMPLE 14 Solution-Phase Assembly of Simple Prototype MorpholinoPolymer, Structural Confirmation, Deprotection, Purification, andAssessment of Binding to Target RNA Sequence

This example describes the preparation, structural confirmation, andassessment of target binding affinity of a simplephosphordiamidate-linked morpholino polymer.

A morpholino hexamer where all Pi moieties are cytosines is assembledfrom dimer prepared as in Example 9D. One third of that dimerpreparation is detritylated (as in Example 12) and the remaining twothirds is activated (as in Example 9D). Half of the activated dimer isreacted with the detritylated dimer to give tetramer, which is purifiedby silica gel chromatography developed with 6% methanol/94% chloroform.The tetramer is detritylated and reacted with the remaining activateddimer to give hexamer, which is purified by silica gel chromatographydeveloped with 10% methanol/90% chloroform.

This phosphordiamidate-linked 5'OH, base-protected hexamer having atrityl moiety on the morpholino nitrogen is designated pd(mC)₆ -trityl.The negative ion Fast Atom Bombardment mass spectrum (3-nitrobenzylalcohol matrix) shows: M-1=2667.9 (100).

This pd(mC)₆ -trityl polymer is next detritylated as in Example 12, andthen a polyethylene glycol 1000 tail is added followed by basedeprotection, as in Example 13. Purification is by cation exchangechromatography followed by desalting on a column of polypropylene, asdescribed in Example 13.

This purified tailed hexamer, pd(mC)₆ -PEG1000, shows an absorptionmaximum at 267.1 nm in neutral aqueous solution, with a calculated molarabsorbance of 42,800. In aqueous solution at pH 1, the same materialshows an absorption maximum at 275.7 nm, with a calculated molarabsorbance of 77,100.

To assess target-binding affinity of the pd(mC)₆ PEG-1000 polymer, 1 mgof polyG RNA (purchased from Sigma Chem Co.) is dissolved in deionizedwater, and 4 volumes of DMSO (spectrophotometric grade from AldrichChem. Co.) is added (stock solution A). Tailed morpholino hexamer,pd(mC)₆ -PEG1000, is dissolved in spectrophotometric grade DMSO (stocksolution B). Phosphate buffer is prepared by adjusting the pH of 0.05NNaOH to 7.4 using phosphoric acid (Buffer C).

Stock solutions A and B are assayed for the actual concentration ofpolymer by UV; the absorbance of stock solution A is measured in 0.1NNaOH and stock solution B is measured in 0.1N HCl. Measurements at thesepH extremes minimize base stacking and other polymer interactions whichcan give absorbance values not proportional to the component monomers.Stock solutions A and B are diluted with Buffer C to give solutions of afinal concentration of 10 micromolar in polymer. The required dilutionsare calculated using molar absorbancies of 65,000 for solution A,poly(G), and 77,100 for solution B, pd(mC)₆ -PEG1000.

Assessment of target binding affinity is carried out in a double-beamscanning spectrophotometer having a temperature-controlled cell housingwhich will accommodate two cells in the reference beam and two in thesample beam.

Using four matched quartz cuvettes, one is filled with 0.5 ml ofpoly(G), 60 micromolar with respect to G monomer, and 0.5 ml of Buffer Cand a second is filled with 0.5 ml of 10 micromolar pd(mC)₆ -PEG1000 and0.5 ml of Buffer C. These two cuvettes are placed in the reference beamof the temperature-controlled cell housing. Next, a third cuvette isfilled with 1 ml of Buffer C and a fourth is filled with 0.5 ml ofpoly(G) 60 micromolar with respect to G monomer and 0.5 ml of 10micromolar pd(mC)₆ -PEG1000. These two cuvettes are placed in the samplebeam of the cell housing. The cell housing is then heated to 50° C. andallowed to cool slowly to 14° C. to assure complete pairing between thepolymer and its DNA target in the fourth cuvette. A scan is then takenfrom 320 nm to 240 nm--which shows a substantial absorbance differencedue to a hypochromic shift in polymer-target mixture, centered around273 nm. The temperature of the cell holder is then raised in 2-degreeincrements to 72° C., with scans taken after each 2-degree rise.

A plot of the absorbance difference as a function of temperature for themorpholino polymer/RNA complex is shown in FIG. 13. The meltingtemperature, Tm, where the complex is half melted, is seen to be 51.5°C. for this morpholino polymer/RNA.

While specific embodiments, methods, and uses of the invention have beendescribed, it will be appreciated that various changes and modificationsof the invention may be made without departing from the invention. Inparticular, although preferred polymer backbone structures have beendescribed and illustrated, it will be appreciated that othermorpholino-based polymers may be constructed according to the backboneconstraints and requirements discussed above.

It is claimed:
 1. A polymer composition comprised of morpholino subunitstructures of the form: ##STR2## where (i) the structures are linkedtogether by uncharged phosphorous-containing, chiral linkages, one tothree atoms long, joining the morpholino nitrogen of one subunit to the5', exocyclic carbon of an adjacent subunit, and (ii) P_(i) is a purineor pyrimidine base-pairing moiety effective to bind by base-specifichydrogen bonding to a base in a polynucleotide.
 2. The composition ofclaim 1, wherein P_(i) is selected from the group consisting of:##STR3## where X is H, CH₃, F, Cl, Br, or I.
 3. The composition of claim1, wherein the linked structures have a form selected from the groupconsisting of: ##STR4##
 4. The composition of claim 1, wherein thelinkage is of the form: ##STR5## where P_(j) is a purine or pyrimidinebase-pairing moiety effective to bind by base-specific hydrogen bondingto a base in a polynucleotide; and,X is F, CH₂ R, O--CH₂ R, S--CH₂ R, orNR₁ R₂ ; and each of R, R₁ and R₂ is H, CH₃, or other moiety that doesnot interfere with said base specific hydrogen bonding.
 5. Thecomposition of claim 1, wherein the linkage is of the form: ##STR6##where P_(j) is a purine or pyrimidine base-pairing moiety effective tobind by base-specific hydrogen bonding to a base in a polynucleotide;and,X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and each of R, R₁ andR₂, is H, CH₃, or other moiety that does not interfere with said basespecific hydrogen bonding.
 6. The composition of claim 1, wherein thelinkage is of the form: ##STR7## where P_(j) is a purine or pyrimidinebase-pairing moiety effective to bind by base-specific hydrogen bondingto a base in a polynucleotide; and,Z is O or S.
 7. The composition ofclaim 1, wherein the linkage is of the form: ##STR8## where P_(j) is apurine or pyrimidine base-pairing moiety effective to bind bybase-specific hydrogen bonding to a base in a polynucleotide; and,Z is Oor S.
 8. The composition of claim 1, wherein the linkage is of the form:##STR9## where P_(j) is a purine or pyrimidine base-pairing moietyeffective to bind by base-specific hydrogen bonding to a base in apolynucleotide; and,X is F, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; andeach of R, R₁ and R₂, is H, CH₃, or other moeity which does notinterfere with said base specific hydrogen bonding.
 9. The compositionof claim 1, wherein the linkage is of the form: ##STR10## where P_(j) isa purine or pyrimidine base-pairing moiety effective to bind bybase-specific hydrogen bonding to a base in a polynucleotide; and,X isF, CH₂ R, O--CH₂ R, S--CH₂ R, or NR₁ R₂ ; and each of R, R₁ and R₂, isH, CH₃, or other moeity which does not interfere with said base specifichydrogen bonding.
 10. The composition of claim 1, which further includesa moiety at one or both termini which is effective to enhance thesolubility of the molecules in aqueous medium.
 11. The composition ofclaim 10, wherein the moiety at one or both termini is polyethyleneglycol.
 12. The composition of claim 1, composed of at least 3morpholino subunits.
 13. The composition of claim 1, wherein at leastone of the P_(i) is a 2,6-diaminopurine.
 14. The composition of claim 1,wherein at least one of the P_(i) is a 5-halouracil.
 15. The compositionof claim 1, wherein at least 70% of the P_(i) are 2-amine containingpurines.